US5037806A - Biologically active method using somatotropins - Google Patents

Biologically active method using somatotropins Download PDF

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US5037806A
US5037806A US06/824,202 US82420286A US5037806A US 5037806 A US5037806 A US 5037806A US 82420286 A US82420286 A US 82420286A US 5037806 A US5037806 A US 5037806A
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bgh
dna
sequence
protein
pgh
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Gwen G. Krivi
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Monsanto Co
Monsanto Technology LLC
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Monsanto Co
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Priority to US06/824,202 priority Critical patent/US5037806A/en
Priority to ES552234A priority patent/ES8801851A1/es
Priority to EP86870024A priority patent/EP0193515B1/de
Priority to NO860658A priority patent/NO300551B1/no
Priority to CA000502495A priority patent/CA1338981C/en
Priority to NZ215260A priority patent/NZ215260A/xx
Priority to AT86870024T priority patent/ATE141327T1/de
Priority to AU53854/86A priority patent/AU596575B2/en
Priority to CN90108714A priority patent/CN1051302A/zh
Priority to DK80886A priority patent/DK80886A/da
Priority to JP61037158A priority patent/JP2583214B2/ja
Priority to DE3650553T priority patent/DE3650553T2/de
Priority to CN86101830A priority patent/CN1021973C/zh
Priority to IL11240386A priority patent/IL112403A/en
Priority to PT82070A priority patent/PT82070B/pt
Priority to IL11240286A priority patent/IL112402A/xx
Priority to HU86742A priority patent/HU210671B/hu
Priority to IL7795086A priority patent/IL77950A/en
Priority to KR1019860001221A priority patent/KR920000849B1/ko
Priority to RO133757A priority patent/RO106956B1/ro
Priority to GR860509A priority patent/GR860509B/el
Priority to IE46786A priority patent/IE80663B1/en
Priority to AU42825/89A priority patent/AU632293B2/en
Priority to SU4830848/13A priority patent/RU2072393C1/ru
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Priority to MX9203728A priority patent/MX9203728A/es
Priority to SU5052718/13A priority patent/RU2094439C1/ru
Priority to LTIP1819A priority patent/LT3916B/lt
Priority to CN94105667A priority patent/CN1100272A/zh
Priority to IL11240395A priority patent/IL112403A0/xx
Priority to JP7128637A priority patent/JP2634390B2/ja
Priority to JP7128636A priority patent/JP2863113B2/ja
Assigned to MONSANTO TECHNOLOGY LLC reassignment MONSANTO TECHNOLOGY LLC ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: PHARMACIA CORPORATION, FORMERLY KNOWN AS MONSATO COMPANY
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/61—Growth hormone [GH], i.e. somatotropin
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K20/00—Accessory food factors for animal feeding-stuffs
    • A23K20/10—Organic substances
    • A23K20/168—Steroids
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K20/00—Accessory food factors for animal feeding-stuffs
    • A23K20/10—Organic substances
    • A23K20/184—Hormones
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K50/00—Feeding-stuffs specially adapted for particular animals
    • A23K50/10—Feeding-stuffs specially adapted for particular animals for ruminants
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K50/00—Feeding-stuffs specially adapted for particular animals
    • A23K50/30—Feeding-stuffs specially adapted for particular animals for swines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/22—Hormones
    • A61K38/27—Growth hormone [GH], i.e. somatotropin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

  • the present invention is directed to a method of expressing heterologous DNA sequences (e.g. eucaryotic genes) in procaryotic organisms.
  • the invention is directed to production in procaryotes of polypeptides having an N-terminal alanine and to various N-alanyl polypeptides that can be produced by that method.
  • valine bGH bovine growth hormone
  • pre-proteins i.e, polypeptides comprised of the mature protein's sequence fused to a leader or signal sequence.
  • Eucaryotic mRNA encodes the entire pre-protein, which is processed after translation to remove the leader sequence and provide the mature protein. While eucaryotic cells are equipped to specifically process such pre-proteins into mature proteins, procaryotic cells are generally not able to recognize the processing signals present in eucaryotic proteins. Thus, if complete complementary DNA (cDNA) transcripts of eucaryotic mRNA are employed as the DNA sequences for expression in procaryotes, the pre-protein, not the mature protein, is found. It is possible to convert pre-proteins to mature proteins in vitro, but not without significant expense.
  • cDNA complementary DNA
  • heterologous DNA is defined herein as DNA at least a portion of which is not normally contained within the genome of the host cell.
  • heterologous DNA include, but are not limited to, viral and eucaryotic genes, gene fragments, alleles and synthetic DNA sequences.
  • heterologous protein or “heterologous polypeptide” is defined herein as a protein or polypeptide at least a portion of which is not normally encoded within the genome of the host cell.
  • the procaryotic control signals include a promoter which signals the initiation of transcription and translation control signals comprising a ribosome binding site, a translation start signal and a translation stop signal. All of these signals except the translation stop signal must be situated in front of the eucaryotic gene or other DNA to be expressed.
  • heterologous DNA e.g. eucaryotic genes
  • the DNA segment encoding the resultant protein is ligated to the DNA encoding all or part of a bacterial protein under the control of its bacterial promoter.
  • the endogenous procaryotic DNA necessarily also contains the ribosome binding site and translation start signal. Expression of such ligated DNA results in what is called a fusion protein comprising the eucaryotic polypeptide linked or fused to a whole or partial bacterial protein. Isolation of the eucaryotic protein may then be achieved by site-specific enzymatic or chemical cleavage at the endogenous-eucaryotic protein fusion site or by selective degradation of the procaryotic polypeptide sequences.
  • coli ⁇ -lactamase European Patent Application No. 95,361 (published Nov. 30, 1983) referring to a fusion protein comprising, in sequence, an endogenous protein at the N-terminus, a translation start signal amino acid, an enterokinase cleavage site, and an exogenous protein (e.g. growth hormone) at the C-terminus.
  • This fusion protein approach is cumbersome in that it requires in vitro processing following purification, and the cost of enzyme(s) for processing commercial quantities can be prohibitive.
  • Fusion proteins have become an attractive system for expressing some eucaryotic genes or other heterologous DNA in procaryotic cells, as the fusion product appears to protect some of the resulting heterologous proteins from intracellular degradation.
  • Bacterial cells appear to recognize some eucaryotic proteins produced therein as foreign and, thus, proceed to degrade these proteins as soon as the proteins are made or shortly thereafter.
  • Fusion proteins engineered for protective purposes can employ endogenous polypeptide sequences at either the amino or carboxy terminus of the heterologous protein.
  • An example of the latter approach is found in European Patent Application No. 111,814 (published June 27, 1984) describing fusion proteins comprising a form of bGH having a synthetic front-end (amino-terminus) and E. coli ⁇ -galactosidase at the C-terminus.
  • the advantages are, again, diminished by the need to subsequently cleave the heterologous protein from the endogenous polypeptide as discussed above.
  • the translation start signal under the control of a bacterial promoter, is located immediately preceding the DNA sequence encoding a heterologous (e.g. eucaryotic) protein free from endogenous protein at both the N-and C-termini of the protein produced.
  • a heterologous protein e.g. eucaryotic
  • the proteins produced by such gene constructs do not require subsequent cleavage to generate the desired protein, they typically include a methionine (in some cases a formyl-methionine) at the N-terminus as the ATG start signal is also a methionine codon.
  • the desired mature protein begins with methionine, the protein will now have an N-terminus altered by inclusion of that methionine residue.
  • N-terminal methionine may be undesirable for several reasons. First, it is possible (although currently believed unlikely) that the methionine may tend to make the protein antigenic in an organism in which the protein without the N-methionine is endogenous. Second, the addition of methionine to the N-terminal portion of the protein may have an undesirable effect on its bioactivity or its physical properties. Third, this altered form of the protein may hinder scientific efforts in determining the relationship of the natural protein's function to its structure. Further, it may be advantageous to have a biosynthetic protein which is structurally as close as possible to a naturally-occurring protein when applying for governmental approval of medical or veterinary applications.
  • Growth hormones also called somatotropins
  • somatotropins are polypeptides produced and secreted by cells of the pituitary gland and are largely species-specific in their actions. In addition to their role in promoting skeletal growth, growth hormones affect a variety of metabolic processes including the stimulation of lactation, increased insulin release from the pancreas and glucagon secretion, and they exert a lipid-mobilizing effect. Exogenous administration of bGH to cattle, for example, has been demonstrated to increase milk production, feed efficiency and/or growth rate, decrease fattening time and increase the lean-to-fat ratio. It is still not fully understood, however, how the hormone exerts these multiple effects.
  • hGH human growth hormone
  • bGH is produced by cattle in multiple forms. Specifically, four forms of bGH are produced which differ at two positions of the protein.
  • the N-terminal amino acid can vary due to a presumed ambiguity in removal of the signal-peptide (leader sequence so that the mature protein begins with either NH 2 -phe-pro or NH 2 -ala-phe-pro.
  • leader sequence so that the mature protein begins with either NH 2 -phe-pro or NH 2 -ala-phe-pro.
  • heterogeneity at amino acid 126 being either a leucine or a valine. This is apparently due to an allelic variation in the bovine population. Wallis (1969) FEBS Letters 3:118-120; Fellows and Rogol (1969) J. Biol. Chem 244:1567-1575: Fernandez et al.
  • the bGH(A,V) and bGH(V) species are sometimes collectively referred to herein as "valine allelic bGH species” or "valine bGH species”.
  • the bGH (A,L) and bGH(L) species are sometimes collectively referred to herein as "leucine allelic bGH species” or "leucine bGH species”.
  • the numbers assigned to the amino acids in the bGH proteins as abbreviated above are for purposes of identification and reference only.
  • pGH porcine growth hormone
  • Pituitary cells of individual cattle have been generally found to contain a mixture of at least bGH(A,L) and bGH(L), or bGH(A,V) and bGH(V).
  • N-terminal analysis of bGH proteins produced in cultured pituitary cells shows an approximately 50:50 mixture of molecules containing either an N-terminal phenylalanine (phe) or an N-terminal alanine (ala).
  • Commercially available preparations, made from pooled pituitaries of many cattle generally include all four molecular forms of pituitary bGH. Furthermore, it has been reported that about thirty percent of the molecules in bGH preparations obtained from pooled glands have valine replacing leucine at position 126.
  • Still another object of the invention is to provide a method for production in procaryotes of eucaryotic or other heterologous polypeptides that have an N-terminal alanine without the need for in vitro processing.
  • Another object of the invention is to produce in procaryotes eucaryotic or other heterologous polypeptides that have the amino acid sequence essentially the same as a naturally-occurring protein which does not have an N-terminal methionine.
  • a further object of the invention is to provide a method of producing in procaryotes polypeptides having the amino acid sequence found in mature eucaryotic polypeptides such as bGH(A,L), bGH(A,V) and pGH(A).
  • the bGH polypeptides produced by methods of the present invention provide a means for potentiating such somatotropin activities as increased milk production, growth rate and/or feed efficiency.
  • a method for bacterially producing a heterologous polypeptide having an N-terminal alanine which comprises causing expression of genomic DNA in selected bacteria, said DNA containing from one to about three contiguous methionine codons including a translation start signal, followed immediately by the codons for said polypeptide, followed by a translation stop signal codon, and recovering the resulting heterologous polypeptide having an N-terminal alanine produced within said bacteria.
  • the present invention provides a method of producing a heterologous polypeptide having an N-terminal alanine comprising causing expression of genomic DNA in selected bacteria, said DNA containing a translation start signal/methionine codon immediately followed by the codons for said polypeptide, followed by a translation stop signal codon, and recovering the resulting heterologous polypeptide having an N-terminal alanine produced within said bacteria.
  • the present invention provides a method for preparing in bacteria a heterologous polypeptide containing an amino acid sequence essentially the same as that of a naturally occurring eucaryotic polypeptide.
  • the present invention provides various compositions comprising such somatotropins as bGH(A,L), bGH(A,V), pGH(A), and mixtures of bGH(A,L) and bGH(A,V), substantially free of polypeptides of bovine or porcine origin, respectively, or of other somatotropin species.
  • compositions comprising a class of valine bGH species enhance milk production in cows to a greater extent than otherwise-identical leucine bGH species or bGH(A,L).
  • compositions able to achieve such enhanced milk production which compositions comprise essentially pure valine bGH species, are provided.
  • Other embodiments include various genes, DNA vectors and transformed bacteria useful in the aforementioned methods, as well as certain methods utilizing the aforementioned compositions for enhancing bovine and/or porcine lactation, pre-adult growth, and/or feed conversion efficiency.
  • the hatched box represents the DNA coding sequence for a bacterial promoter
  • the blackened box represents a heterologous DNA coding sequence
  • the bars represent additional DNA coding sequences as labeled
  • the directional arrow represents the 5' to 3' orientation of the DNA coding sequences. Relevant restriction endonuclease sites are also shown.
  • the DNA regions so marked are for purposes of diagramatic representation only and are not drawn to scale.
  • FIG. 1 depicts the construction of M13mp8/XbaI comprising a M13mp8 vector having inserted therein at the SmaI restriction endonuclease site an XbaI restriction endonuclease site.
  • FIG. 2 depicts the construction of M13mp8/BGH ex-1 comprising M13mp8/XbaI carrying a bGH(L) DNA coding sequence.
  • FIG. 3 depicts the creation of a bGH(A,L) DNA coding sequence by oligonucleotide-directed site-specific mutagensis.
  • FIG. 4 depicts the creation of a bGH(A,V) DNA coding sequence by oligonucleotide-directed site-specific mutagenesis.
  • FIG. 5 depicts the construction of a pMON3209 expression vector comprising pBGH ex-1 carrying a bGH(A,L) DNA coding sequence in place of a bGH(L) DNA coding sequence.
  • FIG. 6 depicts the construction of a pMON3215 expression vector comprising pBGH ex-1 carrying a bGH(A,V) DNA coding sequence in place of a bGH(L) DNA coding sequence.
  • FIG. 7 depicts the construction of M13mp9/PGH ex-1 comprising M13mp9 carrying a pGH(P) DNA coding sequence.
  • FIG. 8 depicts the creation of a pGH(A) DNA coding sequence by oligonucleotide-directed site-specific mutagenesis.
  • FIG. 9 depicts the construction of pBGH ex-1 * comprising pBGH ex-1 wherein the EcoRI restriction endonuclease site located upstream from the 5' end of the ptrp DNA coding sequence has been removed.
  • FIG. 10 depicts the construction of a pMON3213 expression vector comprising pBGH ex-1 * carrying a pPGH(A) DNA coding sequence in place of a bGH(L) DNA coding sequence.
  • the present invention provides a method for producing in a procaryote a heterologous polypeptide, such as a eucaryotic (e.g. mammalian or avian) protein, that has an N-terminal alanine.
  • a heterologous polypeptide such as a eucaryotic (e.g. mammalian or avian) protein
  • the polypeptide is produced without an N-terminal methionine, and the need for in vitro processing to produce such a polypeptide without an N-terminal methionine is eliminated.
  • the consistent production of such a polypeptide lacking an N-terminal methionine present in the gene coding sequence is a novel and quite unexpected result.
  • the present invention provides a valuable method for production of essentially pure proteins that have an N-terminal alanine.
  • proteins include, without limitation, given species of bovine and porcine somatotropin and variants thereof, the plant proteins ribulose-1,5-bisphosphate carboxylase small subunit, glutathione S-tranferase, and heat shock protein 70.
  • the invention is also useful for the production of other polypeptides where it is desirable to have an alanine at the N-terminus rather than a methionine. It can be desirable to have an N-terminal alanine rather than an N-terminal methionine because, inter alia, the N-alanyl form of the polypeptide may be less immunogenic, or have different physical properties or a modified bioactivity.
  • the bGH(A,L), bGH(A,V) and pGH(A) coding sequences were inserted into expression vectors such that the final gene sequence comprised, sequentially, a promoter, a ribosome binding site, an ATG start/methionine codon immediately adjacent the DNA coding sequence for either bGH(A,L), bGH(A,V) or pGH(A), and a translation stop codon.
  • E. coli were then transfected with a given expression vector carrying the desired gene sequence and cultured under conditions which would allow for expression of the desired heterologous DNA and production of the desired heterologous protein. The proteins so produced were then sequenced and assayed for their appropriate biologic activity.
  • the protein recovered from the procaryotic organism actually has alanine at the N-terminus, not a methionine. It is believed that a similar result is obtainable when that alanine codon is immediately preceded by up to about three contiguous methionine codons which include the start signal for translation of the mRNA encoding the desired polypeptide product.
  • the DNA including that translation start signal and the codons for the desired polypeptide product can include any sequence appropriately coding for met ala, met met ala, met met met ala, or any functional equivalent thereof.
  • N-alanyl polypeptide is herein defined as a polypeptide having an alanine at its amino terminus. While applicant does not wish to be bound by the following theory of mechanism, it is believed that subsequent to translation, the N-terminal methionine of the polypeptide is enzymatically removed by the procaryote if the next amino acid is alanine or another amino acid having similar characteristics (e.g. polarity or hydrophobicity) that facilitate such removal of the N-methionine. It is furthermore believed that many procaryotes are capable of removing N-terminal methionines from heterologous and/or endogenous polypeptides when said N-terminal methionines are immediately followed by an alanine.
  • procaryotes e.g. various bacteria known and publicly available through recognized microorganism depositor institutions such as the ATCC or otherwise
  • procaryotes include but are believed not limited to E. coli and various strains thereof. Indeed, it is contemplated that any procaryote capable of producing a polypeptide having an N-terminal alanine from a DNA coding sequence which begins with from one to about three contiguous methionine codons immediately followed by an alanine codon is potentially useful in the practice of the invention disclosed herein.
  • procaryotic organisms may be screened for their ability to produce such N-alanyl polypeptides by inserting into the genome of said organisms a gene comprising sequentially a promoter operative in said organism, DNA encoding a ribosome binding site, from one to about three contiguous methionine codons including a translation start signal followed immediately by the codons for a heterologous N-alanyl polypeptide and a translation stop signal, thereafter causing expression of said gene and then determining the N-terminal amino acid sequence of the heterologous polypeptide so produced.
  • a procaryotic organism has been found to produce internally such a heterologous N-alanyl polypeptide, that organism is within the class defined as " selected" for purposes of the disclosure and claims herein.
  • three different isolates of E. coli strain K12 all of which have been deposited with the American Type Culture Collection, Rockville, Md., and have been assigned accession numbers ATCC 39936, 53010, and 53009 demonstrate an ability to remove N-terminal methionines when said N-terminal methionines are immediately followed by an alanine.
  • the present discovery is significant since it provides a method of producing in procaryotes heterologous polypeptides that have N-terminal alanines.
  • the method of the present invention is employed to generate two bGH species, bGH(A,L) and bGH(A,V), and one pGH species, pGH(A), free from other bovine or porcine proteins and/or other bGH or pGH species, respectively.
  • the method provides for production of bGH(A,L), bGH(A,V) or pGH(A) as single species.
  • the ability to produce a single bGH or pGH species with amino acid sequences homologous to naturally occurring somatotropins has tremendous import for determining the precise bioreactivity of each bGH or pGH species given the numerous potentiating effects of bGH and pGH generally as cited above.
  • valine allelic bGH species result in a greater enhancement in milk production than the leucine allelic bGH species was both surprising and unexpected.
  • This finding further suggests that differences in the relative effectiveness of bGH species in potentiating such other growth hormone properties as increased feed efficiency and growth stimulation can similarly be defined.
  • the bGH species most effective in achieving a specific growth hormone induced response can now be produced.
  • Such other available technologies include, but are not limited to, chemical synthesis of entire bGH proteins or fragments thereof, and/or production in other microorganisms such as yeast or in mammalian cells using known recombinant DNA techniques.
  • somatotropin variants include an altered form of bGH(V) wherein the alteration comprises the addition of a methionine to the amino terminus.
  • This variant produced by genetically transformed bacteria, has also been found to increase lactation in dairy cows to a surprisingly great extent when administered in a lactation-enhancing amount.
  • valine for leucine at position 126 significantly enhances the bioavailability and/or bioreactivity of such somatrotropin protein(s).
  • x-ray crystallography performed on somatotropin proteins indicates that the region of said proteins from about amino acid 90 to about amino acid 135 constitutes a relatively flexible region.
  • flexible regions often comprise a bioreactive site (e.g. a site which interacts with biologic receptors and/or other portions of the same protein to achieve a bioactive form).
  • bGH bGH
  • A,V bGH
  • changes at or around amino acid position 126 can include a substitution of other more hydrophilic and/or smaller amino acids (relative to leucine) providing that the described enhancement in milk production is not diminished to an intolerable degree.
  • valine bGH species containing an N-terminal phe 1 , or an N-terminal ala -1 can achieve the described enhancement in milk production when administered to cows. It is also anticipated that changes in the amino terminus (e.g. met- -1 ) which do not substantially differ from the N-terminus of naturally-occurring bGH would not interfere to an intolerable degree with the ability of valine bGH species to enhance milk production to a measurably greater extent than otherwise-identical leucine bGH species or bGH(A,L).
  • a bGH composition wherein the concentration of valine bGH species, based on the weight of all bGH in the composition, is substantially greater than the concentration of valine bGH species in pooled pituitary bGH preparation would similarly achieve the described result.
  • the present invention is a refinement in the use of a recombinant DNA technology to directly produce in procaryotes heterologous polypeptides.
  • the description of the invention presupposes knowledge of the basic techniques employed in recombinant DNA technology to isolate and clone DNA sequences encoding polypeptides, the rearrangement or altering of cloned DNA sequences, and the expression of cloned or modified DNA sequences in transformed microorganisms.
  • Such techniques are within the skill of the art. (See, e.g., Molecular Cloning: A Laboratory Manual; Maniatis, Fritsch & Sambrook eds. 1982).
  • the DNA sequence coding for the desired heterologous polypeptide to be produced in the procaryote is chosen and isolated, or a DNA sequence that encodes it is constructed or chemically synthesized.
  • the polypeptide is a eucaryotic protein. If the polypeptide is small and the complete amino acid sequence known, a synthetic DNA molecule or sequence encoding the polypeptide can be constructed. If the amino acid sequence of the polypeptide is unknown, or its size is too large for the synthesis of a corresponding DNA sequence to be practical, a cDNA sequence can be prepared by reverse transcription from corresponding mRNA obtained from tissues or cells expressing the polypeptide.
  • the sequence for bGH can be so obtained from bovine pituitaries by now routine procedures described by Goodman et al. Methods in Enzymology 68:75-90 (1979).
  • a cDNA sequence can be prepared from mRNA isolated from cells transformed with genomic DNA isolated from a native gene bank with an appropriate probe.
  • Genomic DNA can also be modified in various vector systems such that the DNA can be expressed in procaryotes. These techniques are within the skill of the art.
  • a heterologous DNA sequence containing the codons for the desired polypeptide it may be desirable to make certain modifications in the nucleotide sequence of the molecule. For example, if the molecule has been produced by reverse transcription from an mRNA template, it will often contain at least a portion of the DNA encoding the leader sequence of the pre-protein. Thus, it is necessary to remove all of the leader-sequence DNA prior to the first codon of the desired protein. In some cases it may be necessary to add or substitute an alanine codon at the beginning of the sequence encoding the desired protein if it does not already have an N-terminal alanine codon.
  • a translation start signal (which is also a methionine codon) is introduced upstream and immediately adjacent to the alanine codon.
  • start signal/methionine codon will usually (and preferably) be the nucleotide sequence ATG, the sequence GTG can also occasionally serve as a start initiation signal/methionine codon.
  • the presence of more than one methionine codon, e.g. two, three or possibly more contiguous codons for methionine is understood to constitute equivalence within the method of the present invention.
  • At least one translation stop signal should be introduced after the codon for the C-terminal amino acid.
  • translation stop signals include the deoxynucleotide triplets TAA, TGA, and TAG.
  • recombinant DNA techniques are applied to construct a recombinant DNA sequence containing, sequentially, a translation start signal/methionine codon, the codons for the desired polypeptide with the N-terminal alanine codon adjacent to the start signal, and at least one translation stop signal adjacent to the codon for the C-terminal amino acid.
  • heterologous DNA sequences For example, if a DNA molecule is available that encodes a polypeptide which is expressed with an N-terminal structure of NH2-met-x-y . . . where x is an amino acid other than alanine, an alanine codon can be inserted between the translation start signal/methionine codon and the codon for x. Alternately, the codon for x may be deleted and an alanine codon inserted in place thereof. Thus, a protein having the N-terminal structure NH2-ala-x-y . . . or NH2-ala-y . . . , respectively, would be produced in the process of the present invention.
  • variant polypeptide is defined herein as having single or multiple amino acid deletions, substitutions and/or additions as compared to the naturally occurring amino acid sequence of a given polypeptide.
  • variants include but are not limited to met-bGH(L) and met-bGH(V) wherein the amino acid sequence of these variant bGH species are identical to the bGH(L) and bGH(V), respectively, produced by bovine pituitary cells except for the presence of an additional methionine at the N-terminus.
  • variant polypeptides are construed as having an amino acid sequence essentially the same as that of a naturally occurring polypeptide as long as the biologic activity is not diminished to an intolerable degree. Creation and expression of variant polypeptides may be desirable in order to achieve increased accumulation, increased protein stability, to facilitate polypeptide purification, and/or in optimizing biological activity.
  • the above modifications of the DNA molecule encoding the desired polypeptide can be accomplished using restriction enzymes, exonucleases, endonucleases, etc. by techniques known in the art.
  • the general techniques of oligonucleotide-directed site-specific mutagenesis can also be employed to effect the above modifications in the structure or sequence of the DNA molecule and are known to those of skill in the art. See e.g., Zoller & Smith (1982) Nuc. Acids Res. 10:6487-6500; Zoller & Smith, (1983) Meth. Enzymol. 100:468:500; Norris et al., (1983) Nuc. Acids Res. 11:5103-5112.
  • any appropriate cloning vector preferably containing a marker function, may be used, for example E. coli plasmid vectors which include Col EI, Hershfield et al., Proc. Nat'l. Acad. Sci. U.S.A.
  • heterologous DNA sequences may be removed from the recombinant vectors and inserted into an expression system for production and isolation of the desired heterologous protein as described more fully below. Modifications of the heterologous DNA sequence, by methods known to those skilled in the art may be made prior to insertion of these DNA sequences into an expression vector or following said insertion.
  • M13mp8 described by Messing et al., Gene (1982) 19:269, modified to contain an XbaI restriction site as shown in FIG. 1, was chosen as a cloning vector along with M13mp9, Id.
  • the M13mp8 and M13mp9 vectors collectively "M13 vectors", allow for isolation of recombinant vectors in both double-stranded (ds) or replicative form (RF) and single-stranded (ss)DNA forms. Isolation of RF DNA recombinant vectors facilitates the subsequent insertion of the replicated desired DNA sequences into expression vectors, as shown in FIGS. 5, 6 and 10.
  • isolation of the single-stranded form of these recombinant vectors facilitates both isolation of recombinant vectors which contain the desired DNA sequence in a proper 5' to 3' orientation for expression and the creation of any DNA sequence modification by such techniques as oligonucleotide-directed site-specific mutagenesis, as shown in FIGS. 3, 4 and 8.
  • these M13 vectors can accommodate DNA fragments or genes up to 4kb in length which insures the cloning of a typical, entire, eucaryotic gene sequence.
  • the marker function employed in the M13 vectors involves the enzyme for ⁇ -galactosidase.
  • the desired heterologous DNA sequence is inserted into the lacZ gene fragment carried on the M13 vector which disrupts the normal complementation of the lacZ gene fragment carried on the M13 vector with the partial lacZ gene fragment carried on the chromosomal DNA of the host cell (e.g. E. coli JM101) so that said host is no longer capable of metabolizing lactose present in the bacterial growth medium.
  • the host cell e.g. E. coli JM101
  • coli infected with M13 vectors which do not have a foreign gene sequence inserted into the vector borne lacZ gene fragment are capable of metabolizing lactose present in the bacterial growth medium and yield characteristic blue plaques when the bacteria are grown on agar containing 1 ⁇ YT medium comprising 0.8% (w/v) tryptone, 0.5% (w/v) yeast extract, 0.5% (w/v) NaCl and a color indicator for ⁇ -galactosidase.
  • the plaque coloration of E. coli infected with recombinant vectors carrying an inserted heterologous DNA sequence in the M13 lacZ gene fragment is clear or colorless when the bacteria are grown on said medium.
  • the ssDNA's of these recombinant vectors were modified by oligonucleotide-directed site-specific mutagenesis to create the DNA coding sequences for bGH(A,V), bGH(A,L), bGH(V) and pGH(A).
  • bGH(L) was modified to add a codon for alanine, e.g. GCC, at the 5'-end of the bGH(L) coding sequence as shown in FIG. 3. It is anticipated that any of the four codons for alanine may be so added.
  • the preferred alanine codon for optimal somatotropin yield in the expression system employed herein is GCC.
  • the bGH(A,V) coding sequence was created by oligonucleotide-directed site-specific mutagenesis of the bGH(A,L) coding sequence as shown in FIG. 4, by converting the leucine codon at amino acid position 127 [in bGH(A,L)] to a valine codon, e.g. GTG. It is again anticipated that any codon for valine may be employed in this conversion.
  • the creation of a bGH(A,V) coding sequence was again confirmed by DNA sequence analysis of the resultant bGH(A,V) coding sequence.
  • the bGH(V) coding sequence which results in production of met-bGH(V) proteins in bacteria transfected with an expression vector comprising the bGH(V) coding sequence was similarly created by oligonucleotide-directed site-specific mutagenesis of the bGH(L) coding sequence by converting the leucine codon at amino acid position 126 [in bGH(L)] to a valine codon, e.g. GTG.
  • heterologous DNA sequences as exemplified for bGH(A,L), bGH(A,V) and pGH(A), these sequences may be replicated and numerous copy numbers generated by propagation of the respective recombinant vectors by methods known to those skilled in the art and referenced above.
  • These heterologous DNA sequences may now be inserted into any appropriate expression vector for production in procaryotes of the desired heterologous polypeptides.
  • an appropriate expression vector should contain the necessary transcription and translation signals for production of a heterologous protein in the chosen host cell along with a marker function for identification of those expression vectors into which the desired heterologous DNA sequence has been inserted.
  • a procaryotic expression vector the recombinant DNA sequences can be added to the genetic complement of a procaryotic organism via transduction, transformation or transfection (collectively referred to herein as "transfection") and said organism can then be cultured under conditions (generally governed by the promoter and host employed) that cause the desired polypeptide to be produced.
  • transfection transformation, transformation or transfection
  • expression vectors pBGH ex-1 see Seeburg et al., DNA (1983) 2(1):37-45, and pBGH ex-1 *, comprising a modified pBGH ex-1 vector, were employed.
  • the bGH ex-1 expression vector is a pBR322 bacterial plasmid carrying a gene for bGH(L).
  • the gene comprises, in sequence, a tryptophan promoter (ptrp), a Shine-Delgarno sequence, an ATG translation start/methionine codon immediately adjacent the N-terminal phenylalanine coding sequence, the first amino acid of the bGH(L) polypeptide, the bGH(L) coding sequence and a translation stop codon.
  • the marker function on the pBGH ex-1 expression vector is antibiotic resistance.
  • pBGH ex-1 carries two antibiotic resistance genes, one for ampicillin (amp r ) and the second for tetracycline resistance (tet r ) which confer specific antibiotic resistance to otherwise susceptible host cells stably transformed with the expression vector.
  • stable transformants may be selected for by growth in media containing either tetracycline, ampicillin or both antibiotics.
  • expression vectors pMON3209 and pMON3215 comprising pBGH ex-1 carrying bGH(A,L) and bGH(A,V) coding sequences, respectively, in place of the bGH(L) coding sequence were generated as shown in FIGS. 5 and 6, respectively.
  • a bacteria such as E. coli, was then stably transformed with one of these expression vectors and transformants selected by growth on media containing the appropriate antibiotic.
  • the expression plasmids contained within the transformed bacteria were then screened for the presence of the bGH(A,L) 3' orientation by restriction enzyme cleavage.
  • pBGH ex-1 * comprising the pBGH ex-1 vector modified by the removal of the EcoRI restriction site located upstream from the 5'-end of the ptrp coding sequence, was employed to create the expression vector pMON3213 carrying the pGH(A) coding sequence in place of the bGH(L) coding sequence.
  • the resultant pBGH ex-1 * vector contains only a single EcoRI site as shown in FIG. 9. The replacement of the pGH(A) coding
  • FIG. 10 sequences for the bGH(L) coding sequence in pBGH ex-1 * to create the expression vector pMON3213 is shown in FIG. 10.
  • E. coli were then transformed with said mixture and transformants selected by growth in antibiotic-containing media.
  • the expression plasmids contained within the transformed bacteria were then screened for the presence of the pGH(A) coding sequences by restriction cleavage.
  • E. coli W3110 E. coli LE392 or E. coli strain 294, deposited and having ATCC accession numbers 39936, 53010, and 53009, respectively, with one of the expression vectors, pMON3209, pMON3215 or pMON3213 in accordance with the method described more fully below.
  • the transformed E. coli W3110 were then cultured under conditions which permit expression of the somatotropin genes and production of the desired heterologous polypeptides.
  • heterologous peptide produced will depend on both the protein and host cell chosen. It has been observed, for example, that the heterologous proteins produced in such bacteria as E. coli are often precipitated within the cell in the form of "refractile" bodies. The term “refractile” is used because these bodies can actually be seen using a phase contrast microscope. A method useful in recovering heterologous proteins, in biologically active form is described in European Patent Application 114,506 (published Aug. 1, 1984) incorporated herein by reference. Briefly, this purification method comprises concentrating the host cells, lysing these cells to generate a cellular extract or homogenate and then isolating the refractile bodies by differential centrifugation, all the steps of which are known to those skilled in the art.
  • the isolated refractile bodies are dissolved in a strong denaturant such as guanidine hydrochloride and the solubilized protein is then exchanged in a suitable solvent (for example, urea), purified by chromatographic means, and finally biologically activated, i.e., permitted to assume its active configuration and then oxidized so that such configuration is maintained by disulfide bonds between its appropriate cysteine residues, as described in European Application 114,506.
  • a suitable solvent for example, urea
  • biologically activated i.e., permitted to assume its active configuration and then oxidized so that such configuration is maintained by disulfide bonds between its appropriate cysteine residues, as described in European Application 114,506.
  • the heterologous peptide expressed by the method of this invention has an N-terminal structure of NH 2 -ala . . . .
  • the remaining polypeptide is typically in the methionyl form, having an N-terminal structure of NH2-met-ala . . . .
  • the somatotropin polypeptide species produced and isolated as described above were shown to exhibit somatotropin-like biological activity as assayed in the rabbit liver receptor assay performed as described by Tsushima and Friesen, J. Clin. Endocrinol. Metab. (1973) 37:334-337 and a rat weight gain bioassay.
  • the bioactivities of the E. coli produced somatotropins are assessed relative to a known lot of somatotropin (e. g. bovine or porcine pituitarg somatotropin) by relating the amount of weight gain of hypophysectomized rats to varying amounts of injected compound.
  • titrated dosages (between 0 and 60 micrograms) of the unknown or standard somatotropin source are injected into hypophysectomized rats (95-135 g) on a daily basis for 7 or more days.
  • body weight gains of animals receiving the known and unknown hormone lots are regressed on log transformed dosages. The slopes are tested to insure nonparallelism and intercept commonality. Bioactivities are reported as the ratio of the slopes times the activity of the standard.
  • N-alanine bGH products of this invention increases milk production by cows and are believed to decrease the amount of feed required for a given output of milk by such cows.
  • Administration to dairy cows of a lactation-enhancing amount of bGH species containing a valine at or around position 126 of the mature bGH protein is particularly well suited for potentiating milk production by these cows.
  • Such products of this invention may be administered to cows by injection, infusion or implantation in polymers or other media known to achieve the delivery of a required dosage in the circulatory system.
  • Pharmaceutically acceptable base formulations such as solutions, emulsions or gels may be used, either encapsulated or not.
  • formulations may contain a single bGH species or variant thereof or any prescribed combination of naturally-occurring and/or variant polypeptides [e.g. a mixture of bGH(A,V) and bGH(A,L) and/or a mixture of bGH(A,V) and met-bGH(A)].
  • Dosages may range from at least about 0.005 mg to about 200 mg per animal per day and preferably from about 5 mg to about 40 mg per animal per day. The amount most effective for increasing milk production and/or feed-to-milk efficiency may be determined by routine experimentation. The actual preferred dosage of bGH is dependent on such variables as the size, general health and nutritional status of the specific animal.
  • milk production can be used to assess the effect of bGH on cows
  • other production properties of the cows such as overall growth rate and meat production can also be employed.
  • bGH can, if desired, be administered to cows with other beneficial agents such as other biologically active proteins, antigens, or the like, and thereby provide enhanced effects.
  • the invention also contemplates the production of variants of bGH which have an N-terminal alanine but which have deletions, additions, inversions and/or substitutions along the polypeptide chain.
  • modifications having desirable lactation and/or growth enhancing properties can be identified by routine testing in cows.
  • oligonucleotides were synthesized in the Department of Biological Sciences, Monsanto, employing an Applied Biosystems DNA synthesizer in accordance with the procedure set forth by the manufacturer, Applied Biosystems, Inc., Foster City, Calif. Restriction enzymes and DNA modifying enzymes were purchased from New England Biolabs (Beverly, Mass.), New England Nuclear (Boston, Mass.) and Bethesda Research Laboratories (BRL) (Gaithersburg, Md.). An XbaI linker was obtained from Collaborative Research, Inc. (Lexington, Mass.). T4 DNA ligase was purchased from BRL (Gaithersburg, Md.).
  • T4 DNA kinase was purchased from New England Biolabs (Beverly, Massachusetts). 32 P-labeled nucleotides were purchased from Amersham (Arlington Heights, Ill.) E. coli DNA polymerase I, Klenow fragment, was purchased from New England Nuclear (Boston, Mass.). E. coli JM101 was obtained from Dr. Jo Messing, University of Minnesota (St. Paul, Minn.).
  • Restriction enzyme digestions, the T4 DNA ligase reactions, and E. coli DNA polymerase I, Klenow fragment, reactions may be carried out in accordance with the procedures set forth by the manufacturers.
  • Preferred buffers for the following restriction enzymers are as follows: for XbaI: 100 mM NaCl, 50 mM tris, pH 7.5, 10 mM MgSO 4 ; for EcoRI, Hind III and Sma I: 50 mM NaCl, 10 mM Tris, pH 7.5, 10 mM MgSO 4 .
  • T4 DNA ligase reactions were run in buffers containing 25 mM Tris, pH 8.0, 10 mM MgCl 2 , 10 mM dithiothritol (DTT), 2 mM spermidine and 0.2 mM ATP.
  • E. coli DNA polymerase I Klenow fragment, was used in a buffer containing 20 mM Tris, pH 7.2, 10 mM MgCl 2 , 10 mM (DTT), 1 mM ATP, and 1 mM each dATP, dGTP, dCTP, dTTP.
  • Alpha- 32 P-dATP 400 ci/mmol was added to the Klenow reaction if labeling of the newly synthesized DNA strand was desired.
  • Oligonucleotides were labeled using gamma- 32 P-ATP (sp. act. greater than 5,000 ci/mmol) and T4 DNA kinase in 100 mM Tris, pH 8.0, 10 mM MgCl 2 , 5 mM DTT.
  • Plasmids carrying DNA coding sequences for bGH(L) and pGH(P) were obtained from Genentech, Inc., So. San Francisco, Calif. These plasmids can be prepared as described in European Patent Application 75,444 (published Mar. 30, 1983); Seeburg et al., DNA (1983) 2(1):37-45; Goeddel et al. Nature (1979) 281:544-548; DeBoer et al., in Promoters: Structure and Function (1982); M. J. Chamberlin and R. Rodriguez eds., Chapter 293; Miozzari and Yanofsky, J. Bacteriol.
  • the first 21 translated codons in the DNA for bGH(L) are ATG TTC CCA GCT ATG TCT CTA TCT GGT CTA TTC GCT AAC GCT GTT CTT CGT GCT CAG CAT CTT or functional equivalents thereof. (Functional equivalents of those codons can of course be used alternatively.)
  • Other relevant portions of these publications are incorporated herein by reference.
  • M13mp8 and M13mp9 were obtained from Dr. Jo Messing, University of Minnesota (St. Paul, Minn.).
  • the following example demonstrates the construction of three DNA coding sequences which, when expressed, provide for the direct production in bacteria of polypeptides containing an N-terminal alanine.
  • DNA coding sequences were constructed such that the translation initiation start/methionine codon (ATG) is immediately followed by an alanine codon (e.g. GCC).
  • the three DNA coding sequences comprising bGH(A,L), bGH(A,V) and pGH(A), were constructed by oligonucleotide-directed site-specific mutagenesis of previously isolated somatotropin DNA sequences.
  • This example also demonstrates the construction of a bGH(V) DNA coding sequence which when expressed in bacteria causes production of met-bGH(V).
  • the somatotropin, bGH(L), DNA coding sequence was excised from pGH ex-1 as an XbaI fragment and cloned into the XbaI site of a modified M13mp8 vector (M13mp8/XbaI). Construction of the M13mp8/XbaI vector which contains an XbaI linker in the original SmaI site is shown in FIG. 1. As shown in FIG. 2, XbaI cleaves at either end of the bGH(L) DNA coding sequence thus excising the complete bGH(L) coding sequence.
  • the XbaI restricted pBGH ex-1 plasmid was mixed in the presence of T4DNA ligase with RF M13mp8/XbaI DNA linearized by XbaI restriction enzyme cleavage and treated with calf intestine alkaline phosphatase. The mixture was then incubated overnight at 14° C. Treatment with calf intestine alkaline phosphatase prevents recircularization of the M13mp8/XbaI vector. Insertion of the bGH(L) DNA coding sequence into the M13mp8/XbaI vector to create recombinant vector M13mp8/B was initially ascertained by colorless plaque formation on a lawn of bacteria, E.
  • Insertion of the bGH(L) coding sequence was confirmed by cleavage of isolated RF DNA, Molecular Cloning: A Laboratory Manual, Maniatis, Fritsch & Sambrook, eds. (1982), Chapter 3, of the recombinant vector with XbaI which yields a 590 base pair fragment comprising the inserted sequence.
  • the 590 base pair fragment was identified by agarose gel electrophoresis in one percent (w/v) agarose as described in Molecular Cloning: A Laboratory Manual, Maniatis, Fritsch & Sambrook, eds. (1982). All subsequent restriction fragments were identified by this referenced method.
  • the orientation of the inserted bGH(L) coding sequences was ascertained by cleavage of the RF recombinant vector with SmaI and HindIII. When the coding sequences are in the correct 5' to 3' orientation, cleavage with these restriction enzymes should yield a 207 base pair fragment.
  • the isolation of ss phage DNA was conducted in accordance with the method of Messing et al., Gene (1982) 19:269.
  • the M13mpS/B vector was then employed as a template in the oligonucleotide-directed site-specific mutagenesis essentially as described by Zoller and Smith, Nuc. Acids Res. (1982) 10:6487-6500, Zoller and Smith Methods in Enzymol. (1983) 100:468-500.
  • Norris et al., Nuc. Acid Res. (1983) 11:5103-5112 the relevant portions of which are herein incorporated by reference.
  • FIG. 3 diagrams the mutagenesis procedure for creation of a DNA coding sequence for bGH(A,L) from the bGH(L) coding sequence.
  • an oligonucleotide primer (see Table 1, below) containing the sequence of the desired mutation is used to prime synthesis of a closed-circular DNA copy of the ssDNA M13mp8/BGH ex-1 template.
  • the closed-circular dsDNA molecules thus generated are separated from incomplete and ssDNA circles by alkaline sucrose gradient centrifugation as described by Zoller and Smith, Methods in Enzymol. (1983) 100:468-500.
  • the closed-circular dsDNA molecules are then used to transform E.
  • M13mp8/bGH ex-1 phage M13mp8/bGH ex-1 phage.
  • a typical filter washing protocol employed a room temperature wash in 6 ⁇ SSC (0.9M NaCl and 0.09M NaCitrate) for ten minutes followed by a 50° C. wash in 6 ⁇ SSC for five minutes and subsequent washings at temperatures increasing by 5° C. Plaques which hybridized to radiolabeled oligonucleotide primer at temperatures higher than the control phages were presumed to carry the newly created bGH(A,L) coding sequence and were termed potential positives. Alternatively, individual colorless plagues were picked from the E.
  • Phage DNA prepared in accordance with Messing et al., Gene (1982) 19:269, was then spotted onto nitrocellulose, hybridized with radiolabeled primer, and washed in increasing temperatures as described above. Phage DNA which showed hybridization temperatures higher than M13mp8/bGH ex-1 control plaques were similarly termed potential positives.
  • bGH(A,V) DNA coding sequence Construction of the bGH(A,V) DNA coding sequence, screening and sequence confirmation were carried out using the same procedure as above except that the template was M13mp8/BGH ex-1 (ala), as shown in FIG. 4, and the oligonucleotide primer was as shown in Table 1, below.
  • the frequency of conversion of the leucine codon to a valine codon was about 10%.
  • bGH(V) DNA coding sequence Construction of the bGH(V) DNA coding sequence, screening and sequence confirmation were carried out using the same procedure as above. Specifically, the template was M13mp8/BGH ex-1 and the oligonucleotide primer was the same as employed to create bGH(A,V). The frequency of conversion of the leucine codon to a valine codon was, again, about 10%.
  • Oligonucleotide-site-specific mutagenesis was similarly employed to add an alanine codon to the pGH(P)DNA coding sequence described by Seeburg et al., DNA (1983) 2(1):37-45.
  • the mutagenesis procedure as diagramed in FIG. 8 was conducted as follows.
  • the 590 base pair pGH(P) DNA coding sequence carried on the pPGH ex-1 plasmid was removed from the plasmid by restriction enzyme cleavage with EcoRI and HindIII which cleave the plasmid at the 5'-and 3'-ends of the pGH(P) coding sequence, respectively, as shown in FIG. 7.
  • the restricted pPGH ex-1 plasmid was then mixed with M13mp9 RF DNA similarly cleaved with EcoRI and HindIII but additionally pretreated with calf intestine alkaline phosphatase to prevent religation of the M13mp9 restriction fragments. T4 DNA ligase was then added to said mixture.
  • E. coli JM101 were then transformed, as described above for M13mp8/BGH ex-1 , with the recombinant M13mp9/PGH ex vector carrying the pGH(P) DNA coding sequence.
  • the transformed E. coli JM101 were then grown on 1 ⁇ YT medium containing colorimetric reagents and selected by colorless plague formation as described previously.
  • Insertion of the pGH(P) DNA coding sequences was confirmed as follows. Colorless plaques were picked and RF M13mp9/PGH ex-1 DNA, isolated as previously described, was then cleaved with EcoRI and Hind III and subjected to agarose gel electrophoresis yielding a 590 base pair fragment comprising the inserted pGH(P) DNA. M13mp9/PGH ex-1 phage were then propagated in E. coli JM101 and the ss phage DNA isolated as described above.
  • the M13mp9/PGH ex-1 DNA was then employed as a template for the oligonucleotide-site-specific mutagenesis, shown in FIG. 8, in accordance with the procedure described above for creation of a bGH(A,L) coding sequence, employing a specific primer (see Table 1, below).
  • the frequency of addition of an alanine codon, herein GCC, to the pGH(P) coding sequence was approximately 12%.
  • the resultant pGH(A) coding sequence was again confirmed by DNA sequence analysis.
  • This example demonstrates the construction and expression of three recombinant expression vectors which provide for the direct production in bacteria of poly peptides containing an N-terminal alanine.
  • the three polypeptides so produced are the somatotropin species bGH(A,L), bGH(A,V) and pGH(A).
  • This example also demonstrates the construction and expression of bGH(V) and expression of bGH(L).
  • the polypeptides so produced are met-bGH(V) and met-bGH(L), respectively.
  • Expression plasmid pBGH ex-1 was also digested with XbaI and subsequently treated with calf intestine alkaline phosphatase to prevent religation of the restriction framents. Each of the digested RF DNA's were then separately mixed with the digested and treated pBGH ex-1 DNA and ligated as described previously overnight at 14° C.
  • the recombinant expression vectors thus formed were labeled pMON3209, pMON32115 and pMON3214 carrying the bGH(A,L), bGH(A,V), and bGH(V) DNA coding sequences, respectively.
  • coli W3110 were then transformed with the ligation mixture containing pMON3209 or pMON32115 or pMON3214 or pBGH ex-1 and grown in Lauria Broth (LB) comprising 1% (w/v) tryptone, 0.5% (w/v) yeast extract and 0.5% (w/v) NaCl and containing 12.5 % ⁇ g/ml tetracycline and 200 ⁇ g/ml ampicillin.
  • E. coli W3110 containing pMON3209 have ATCC accession number 53024.
  • E. coli W3110 containing pMON32115 have ATC accession number 3022. Transformation was conducted, briefly, as follows. Approximately 50 ml of E.
  • Resistant colonies were then selected and the pMON3209, pMON32115, pBGH ex-1 and pMON3214 expression vector DNA's isolated in accordance with the procedure described in Molecular Clonings: A Laboratory Manual, Maniatis, Fritsch & Sambrook, eds. (1982).
  • the pMON3209, pMON32115, pBGH ex-1 and pMON3214 DNA's were screened for the presence of a 590 base pair XbaI fragment and a 200 base pair Hind III/SmaI fragment showing the presence of the bGH(A,L), bGH(V) and bGH(A,V) DNA coding sequences in the correct orientation.
  • the pMON3209 and pMON32115 expression plasmids were further screened by restriction analysis with HaeIII to confirm the presence of a new HaeIII site resulting from the addition of a GCC(alanine) codon. Finally, the 590 base pair XbaI fragment from the pMON3209, pMON3214 and pMON32115 vectors were partially sequenced as described previously to confirm the presence of the bGH(A,L), bGH(V) and bGH(A,V) DNA coding sequences in these expression vectors.
  • 0.5 ml of the overnight cultures were then used to separately inoculate 25 ml of M9 media comprising (per liter of media) 100 ml 10 ⁇ salts (70 grams (g) Na 2 HPO 4 , 30g KH 2 PO 4 , 5 g NaCl, 10 g NH 4 Cl per 1000 ml total volume) 1.2 ml 1M MgSO 4 , 0.25 ml 0.1% B 1 , 12.5 ml 20% (w/v) glucose, 0.025 ml 1M CaCl 2 , supplemented with 0.5% (w/v) casamino acids and 6.25 ug/ml tetracycline, contained within 250 ml flasks.
  • An aliquot of 0.2 ml was next removed from each of said flasks and individually lysed in sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) buffer and assayed by SDS-PAGE in accordance with Laemmli, Nature (1970) 227:680-685. Proteins of 22,000 daltons were at high levels in E. coli W3110 from both of the two gene constructs.
  • SDS sodium dodecyl sulfate
  • PAGE polyacrylamide gel electrophoresis
  • both 22,000 dalton proteins bound in Western blot assays (see Krivi and Rowold, Hybridoma (1984) 3:252-262) to a monoclonal antibody, F11-A1-B6, (see Id.) produced against bovine pituitary somatotropin, thereby confirming that the polypeptides are related to pituitary somatotropin.
  • E. coli W3110 cells carrying the parental pBR322 plasmid do not produce a protein of 22,000 daltons that binds to anti-somatotropin antibodies.
  • the cells from each flask were then harvested by centrifugation at 6000 ⁇ g at 4° C. for 5 minutes.
  • the pellets were individually resuspended in 12 ml LB plus 7.5% (v/v) DMSO and immediately frozen on dry ice in 1 ml aliquots.
  • the cells were then stored in a liquid nitrogen freezer. Additionally, about 10 micrograms of purified plasmid DNA was stored at -80° C.
  • the pGH(A) coding sequence carried on the recombinant vector M13mp9/PGH ex-1 (ala) was used to replace the bGH(L) DNA coding sequence carried on a modified pBGH ex-1 expression vector.
  • the modified expression vector, pBGH ex-1 * comprises a pBGH ex-1 expression vector wherein the EcoRI restriction site located upstream from the tryptophan promoter (ptrp) has been removed (see FIG. 9).
  • the modified pBGH ex-1 * expression vector was created by partial EcoRI digestion of pBGH ex-1 * followed by SI nuclease treatment to remove the sticky ends.
  • the restricted pBGH ex-1 vector was then recircularized by use of T4 DNA ligase and used to transform E. coli JM101, all as previously described. Plasmid DNA from single colonies was next screened for a 590 base pair EcoRI/HindIII fragment carrying the pGH(A) coding sequence and a 1050 base pair EcoRI/PstI fragment carrying the ptrp sequence (See FIG. 9). Removal of this EcoRI restriction site facilitates the site specific insertion of the pGH(A) coding sequence into the pBGH ex-1 * expression vector in the proper orientation as shown in FIG. 10. The recombinant expression vector thus formed is hereinafter referred to as pMON3213.
  • E. coli W3110 containing pMON3213 have ATCC accession number 53023.
  • Replacement of the pBGH(L) coding sequence with a pGH(A) coding sequence in the pBGH ex-1 * expression vector was confirmed by isolating pMON3213 DNA and then cleaving said expression vector with EcoRI and Hind III to generate a 590 base pair fragment and cleavage with Hae III to show the presence of an additional Hae III restriction fragment generated by the presence of an alanine codon in the pGH(A) DNA coding sequence.
  • Final confirmation of the presence of the pGH(A) DNA coding sequence in the pMON3213 expression vector was made by partially sequencing the EcoRI/Hind III 590 base pair fragment as previously described.
  • E. coli W3110 carrying the pMON3213 expression vector were stored as previously described and employed in large scale (10 to 100 liter fermentation) production of pGH(A) also as previously described.
  • the pGH(A) protein content in the 100-liter batch fermentation was, again, approximately 1 g/liter of broth by radioimmunoassay of Rosner et al., J. Immunol. Methods (1982) 52:175-181.
  • This example was performed in order to determine the N-terminal amino acid sequence of the heterologous proteins, bGH(A,L), bGH(A,V), met-bGH(V), met-bGH(L) and pGH(A), produced in bacteria as examples of the method of the present invention.
  • the somatotropin polypeptides produced in E. coli were purified from crude solubilized refractile bodies containing either bGH(A,L), bGH(A,V), met-bGH(V), met-bGH(L) or pGH(A) by immunoadsorbent chromatography as described by Krivi & Rowold, Hybridoma (1984) 3:151-161. All three somatotropin species purified by immunoadsorbent chromatography appeared to be greater than 95% pure by SDS-PAGE analysis of 1 ug purified protein run on a 7.5 to 15% (w/v) gradient gel performed in accordance with the method of Laemmli, Nature (1970) 227:680-685. Protein concentrations of the purified bGH species were determined by routine high performance liquid chromatography analysis.
  • Proteins purified by immunoaffinity chromatography for use in N-terminal sequence analysis were dialyzed exhaustively against water and then lyophilized. Prior to N-terminal sequence analysis, the purified proteins were resuspended in ammonium bicarbonate buffer comprising 50 mM ammonium bicarbonate plus 0.1% (w/v) SDS and dialyzed against the same buffer to remove residual tris(hydroxymethyl) amino methane(tris) and glycine.
  • An Applied Biosystems Protein Sequencer Model 470A (Applied Biosystems, Inc. Foster City, CA) was then employed for all N-terminal sequencing analysis, as described by Hunkapiller et al. (1983), Methods in Enzymol. 91:399-413 and Hunkapiller et al. (1983), Methods in Enzymol. 91:486-493.
  • Table 2 shows the results of the sequence analysis for several preparations of bGH(A,L), bGH(A,V), met-bGH(V), met-bGH(L) and pGH(A) polypeptides.
  • the amount of protein with N-terminal methionine is shown in the table as a percentage of the total somatotropin in the sample.
  • Two methods were used for methionine quantification. With the indirect method the amount of NH2-met-ala-phe . . . in a predominantly NH2-ala-phe . . . population was calculated from differences in lag signals.
  • the direct method comprising an Edman degradation reaction compared signal strength of PTH-met to PTH-ala after separation of PTH-met from chemical noise by high performance liquid chromatography (HPLC).
  • PTH refers to phenyl-thiohydantoin.
  • the Edman degradation sequencing reaction consists of reacting the N-terminal amino acid with a reagent that causes cleavage of that amino acid and its subsequent release as a PTH-derivative of that amino acid. The latter procedure will give good estimates of g met-ala-phe . . . as long as free amino acid contamination is low.
  • test somatotropins All test somatotropins and control samples were administered parenterally by intramuscular injection into the semitendinosus muscle daily for 21 consecutive days. The individual milk production of each cow was recorded at each PM and AM milking beginning six days prior to the first daily injection through day 21 of injection.

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US06/824,202 US5037806A (en) 1985-02-22 1986-02-04 Biologically active method using somatotropins
ES552234A ES8801851A1 (es) 1985-02-22 1986-02-20 Un metodo para producir bacterianamente un polipeptido heteralogo que tiene actividad somatotropinica.
GR860509A GR860509B (en) 1985-02-22 1986-02-21 Biologically active proteins and a method for their production
IL11240286A IL112402A (en) 1985-02-22 1986-02-21 Methods for producing N-alanyl porcine somatotropin and compositions thereof
RO133757A RO106956B1 (ro) 1985-02-22 1986-02-21 METODã DE SPORIRE A LACTA¦IEI LA VACI
CA000502495A CA1338981C (en) 1985-02-22 1986-02-21 Biologically active proteins and a method for their production
NZ215260A NZ215260A (en) 1985-02-22 1986-02-21 Method of expressing eucaryotic genes in procaryotic organisms
AT86870024T ATE141327T1 (de) 1985-02-22 1986-02-21 Verfahren zur herstellung biologisch aktiver rinder- und schweine-wachstumshormone und mischung mit biologisch aktivem schweine- wachstumshormon
AU53854/86A AU596575B2 (en) 1985-02-22 1986-02-21 Biologically active protein compositions and a method of use
CN90108714A CN1051302A (zh) 1985-02-22 1986-02-21 增加奶牛乳汁分泌的方法
DK80886A DK80886A (da) 1985-02-22 1986-02-21 Biologisk aktive proteiner samt fremgangsmaade til deres fremstillingog anvendelse
JP61037158A JP2583214B2 (ja) 1985-02-22 1986-02-21 牛ソマトトロピン含有組成物およびこれを用いる牛の乳汁分秘促進法
DE3650553T DE3650553T2 (de) 1985-02-22 1986-02-21 Verfahren zur Herstellung biologisch aktiver Rinder- und Schweine-Wachstumshormone und Mischung mit biologisch aktivem Schweine-Wachstumshormon
CN86101830A CN1021973C (zh) 1985-02-22 1986-02-21 有生物活性的蛋白质与其生产的方法
IL11240386A IL112403A (en) 1985-02-22 1986-02-21 A method for producing mature growth hormone from cattle at the end of which amine alanine in E.COLI
PT82070A PT82070B (pt) 1985-02-22 1986-02-21 Metodo para a preparacao de proteinas biologicamente activas
EP86870024A EP0193515B1 (de) 1985-02-22 1986-02-21 Verfahren zur Herstellung biologisch aktiver Rinder- und Schweine-Wachstumshormone und Mischung mit biologisch aktivem Schweine-Wachstumshormon
HU86742A HU210671B (en) 1985-02-22 1986-02-21 Method for production of derivates of grocoth hormon
IL7795086A IL77950A (en) 1985-02-22 1986-02-21 Method and preparations for increasing milk production in cattle
KR1019860001221A KR920000849B1 (ko) 1985-02-22 1986-02-21 생물학적으로 활성인 단백질의 제조방법
NO860658A NO300551B1 (no) 1985-02-22 1986-02-21 Fremgangsmåte for bakteriell fremstilling av et polypeptid, preparat som omfatter et slikt, samt rekombinant DNA-vektor
IE46786A IE80663B1 (en) 1985-02-22 1986-02-24 Method for obtaining biologically active bovine and porcine growth hormone and composition comprising biologically active porcine growth hormone
AU42825/89A AU632293B2 (en) 1985-02-22 1989-10-13 A method for production of biologically active proteins
SU4830848/13A RU2072393C1 (ru) 1985-02-22 1990-08-20 Способ получения рекомбинантного полипептида со свойствами свиного гормона роста
MX9203728A MX9203728A (es) 1985-02-22 1992-06-29 Proteinas biologicamente activas y metodo para su produccion.
SU5052718/13A RU2094439C1 (ru) 1985-02-22 1992-08-03 Рекомбинантный полипептид pgh(a) с n-концевым аланином
LTIP1819A LT3916B (en) 1985-02-22 1994-01-28 Method for production porcine growth hormone
CN94105667A CN1100272A (zh) 1985-02-22 1994-05-10 促进猪成年前生长和提高猪饲料转化效率的组合物和方法
IL11240395A IL112403A0 (en) 1985-02-22 1995-01-20 Method for producing a heterologous polypeptide
JP7128637A JP2634390B2 (ja) 1985-02-22 1995-05-26 アミノ末端アラニンをもつ牛又は豚ソマトトロピンの製法
JP7128636A JP2863113B2 (ja) 1985-02-22 1995-05-26 豚ソマトトロピンを含有する家畜の成長制御用組成物

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US5130422A (en) * 1988-08-29 1992-07-14 Monsanto Company Variant somatotropin-encoding DNA
US5141922A (en) * 1985-02-22 1992-08-25 Monsanto Company Biologically active proteins and a method for their use
US5164482A (en) * 1989-09-13 1992-11-17 Bayer Aktiengesellschaft Recombinant aprotinin variants genetically engineered process for the microbial preparation of homogeneously processed aprotinin variants and the therapeutic use thereof
US5244882A (en) * 1987-03-12 1993-09-14 Amgen, Inc. Porcine growth hormone analogs, and compositions
US5356877A (en) * 1987-03-12 1994-10-18 Amgen, Inc. Methods for promoting growth using porcine growth hormone analogs
US5744328A (en) * 1985-02-22 1998-04-28 Monsanto Company Production of proteins in procaryotes
US5767080A (en) * 1996-05-01 1998-06-16 Cargill, Incorporated Enhanced milk production in dairy cattle
US5951972A (en) * 1990-05-04 1999-09-14 American Cyanamid Company Stabilization of somatotropins and other proteins by modification of cysteine residues
US6692941B1 (en) 1980-08-26 2004-02-17 The Regents Of The University Of California Bovine growth hormone
US20050106670A1 (en) * 2000-03-27 2005-05-19 Applera Corporation Isolated human G-protein coupled receptors that are members of the aminergic subfamily, nucleic acid molecules encoding human GPCR proteins, and uses thereof
US7101981B1 (en) 1980-08-26 2006-09-05 Regents Of The University Of California Bovine growth hormone recombinantly produced in E. coli

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AU599922B2 (en) * 1985-09-10 1990-08-02 Natinco Nv Improving carcass quality
IL82017A (en) 1986-03-27 1996-01-19 Monsanto Co Increased production of protein in bacteria by using a ribosomal linker site based on bacteriophage T7 gene 01
GB8701848D0 (en) * 1987-01-28 1987-03-04 Celltech Ltd Polypeptides
US4863736A (en) 1987-03-16 1989-09-05 Monsanto Company Somatotropin prolonged release
ATE152176T1 (de) * 1988-02-17 1997-05-15 Upjohn Co Verfahren zum regeln des norleucingehalts in polypeptiden
US5089473A (en) * 1988-08-29 1992-02-18 Monsanto Company Somatotropin variants and their use
EP0482124B1 (de) * 1989-07-10 1993-12-22 The Upjohn Company Somatotropinanaloge
US5663305A (en) * 1989-07-10 1997-09-02 The Upjohn Company Somatotropin analogs
US6583115B1 (en) 1989-10-12 2003-06-24 Ohio University/Edison Biotechnology Institute Methods for treating acromegaly and giantism with growth hormone antagonists
US6787336B1 (en) 1989-10-12 2004-09-07 Ohio University/Edison Biotechnology Institute DNA encoding growth hormone antagonists
US5350836A (en) * 1989-10-12 1994-09-27 Ohio University Growth hormone antagonists
US5958879A (en) * 1989-10-12 1999-09-28 Ohio University/Edison Biotechnology Institute Growth hormone receptor antagonists and methods of reducing growth hormone activity in a mammal
GB9803424D0 (en) * 1998-02-18 1998-04-15 Pfizer Ltd Performance enhancement
CN1322211A (zh) * 1998-10-05 2001-11-14 武田药品工业株式会社 除去n－末端蛋氨酸的方法
CN101665788B (zh) * 2009-09-21 2011-11-09 山西大学 人工合成猪生长激素基因及其表达纯化方法
CN101907626A (zh) * 2010-07-20 2010-12-08 广东省农业科学院兽医研究所 一种猪饲料中猪源蛋白的检测方法

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Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7101981B1 (en) 1980-08-26 2006-09-05 Regents Of The University Of California Bovine growth hormone recombinantly produced in E. coli
US6692941B1 (en) 1980-08-26 2004-02-17 The Regents Of The University Of California Bovine growth hormone
US5744328A (en) * 1985-02-22 1998-04-28 Monsanto Company Production of proteins in procaryotes
US5141922A (en) * 1985-02-22 1992-08-25 Monsanto Company Biologically active proteins and a method for their use
US6210928B1 (en) 1985-02-22 2001-04-03 Monsanto Company Production of proteins in procaryotes
US5244882A (en) * 1987-03-12 1993-09-14 Amgen, Inc. Porcine growth hormone analogs, and compositions
US5356877A (en) * 1987-03-12 1994-10-18 Amgen, Inc. Methods for promoting growth using porcine growth hormone analogs
US5130422A (en) * 1988-08-29 1992-07-14 Monsanto Company Variant somatotropin-encoding DNA
US5164482A (en) * 1989-09-13 1992-11-17 Bayer Aktiengesellschaft Recombinant aprotinin variants genetically engineered process for the microbial preparation of homogeneously processed aprotinin variants and the therapeutic use thereof
US5951972A (en) * 1990-05-04 1999-09-14 American Cyanamid Company Stabilization of somatotropins and other proteins by modification of cysteine residues
US6010999A (en) * 1990-05-04 2000-01-04 American Cyanamid Company Stabilization of fibroblast growth factors by modification of cysteine residues
US5767080A (en) * 1996-05-01 1998-06-16 Cargill, Incorporated Enhanced milk production in dairy cattle
US20050106670A1 (en) * 2000-03-27 2005-05-19 Applera Corporation Isolated human G-protein coupled receptors that are members of the aminergic subfamily, nucleic acid molecules encoding human GPCR proteins, and uses thereof

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